RRC ID 31915
著者 Okeke CN, Tsuboi R, Kawai M, Yamazaki M, Reangchainam S, Ogawa H.
タイトル Reverse transcription - 3' rapid amplification of cDNA ends-nested PCR of ACT1 and SAP2 mRNA as a means of detecting viable Candida albicans in an in vitro cutaneous candidiasis model.
ジャーナル J Invest Dermatol
Abstract The presence of viable cells of Candida albicans, in broth or in a reconstructed living skin equivalent, was determined by the detection of amplicons of partial mRNA sequences of the genes encoding fungal actin (ACT1) and secreted aspartyl proteinase 2 (SAP2). The mRNA of both genes were amplified by reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction. Single bands of ACT1 (315 bp) and SAP2 (162 bp) mRNA were amplified from total RNA extracts of C. albicans grown in yeast carbon base-albumin broth or in living skin equivalent tissue; only the former was amplified from Sabouraud broth-grown organisms. Primer pairs targeted for ACT1 and SAP2 were Candida genus-specific and C. albicans-specific, respectively. The sensitivity limits of the assay were 100 fg of total RNA or 10 cells of C. albicans, by ethidium bromide staining. When C. albicans-infected living skin equivalent was exposed to amorolfine, amplicons of ACT1 and SAP2 mRNA were not detected in total RNA extracts. Non-amplification of the mRNA correlated with the absence of C. albicans growth in Sabouraud agar cultures of living skin equivalent samples. Reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction of the mRNA encoding specific proteins of an organism has potential application in determining the viability of the organism in tissue, thus monitoring the efficacy of an antimicrobial therapy, and in detecting mRNA expressed in very little amounts in tissue.
巻・号 114(1)
ページ 95-100
公開日 2000-1-1
DOI 10.1046/j.1523-1747.2000.00845.x
PII S0022-202X(15)40737-7
PMID 10620122
MeSH Actins / genetics* Aspartic Acid Endopeptidases / genetics* Candida albicans / isolation & purification* Candida albicans / physiology Candidiasis / metabolism* Cells, Cultured DNA, Complementary / genetics* Dermatomycoses / microbiology* Fibroblasts / microbiology Fungal Proteins* Humans Keratinocytes / microbiology RNA, Messenger / genetics* Reverse Transcriptase Polymerase Chain Reaction Sensitivity and Specificity Skin, Artificial
IF 7.143
引用数 8
WOS 分野 DERMATOLOGY
リソース情報
病原微生物 IFM 45828 IFM 47625